GSM7892758: Young_1; Mus musculus; RNA-Seq
Retinas were digested using the Neurosphere dissociation kit (Miltenyi Biotec),  vascular structures were pelleted down, supernatant discarded and the pellet digested again and triturated by pipetting. Cell suspension was then washed with HBSS/1% BSA (wt/vol)/0.1% D-glucose (wt/vol), and filtered through a 40um strainer. CD31+ cells were enriched using CD31 microbeads and the autoMACS Pro-separator (Miltenyi Biotec) for magnetic separation. Sorted cells were then washed, counted, and a suspension of 1000 cells/ul was loaded onto the microfluidic chip and libraries prepared using the 10x Genomics v3.1 kit. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.